Generation of Myomaker−/− mice
The Myomaker mouse strain used for this research project was created from ES cell Tmem8c clone (EPD0626_5_C12) obtained from KOMP Repository (www.KOMP.org
) and generated by the Wellcome Trust Sanger Institute29
. This clone was injected into 3.5-day-old C57BL/6 blastocysts by the Transgenic Core Facility at University of Texas Southwestern Medical Center. High-percentage chimeric male mice were bred to C57BL/6 females to achieve germline transmission of the targeted allele. Myomaker+/−
mice were intercrossed to generate Myomaker−/−
mice. All experimental procedures involving animals in this study were reviewed and approved by the University of Texas Southwestern Medical Center’s Institutional Animal Care and Use Committee.
Quantitative real-time PCR (qPCR)
Total RNA was extracted from either mouse tissue or cultured cells with TRIZOL (Invitrogen) and cDNA synthesized using Superscript III reverse transcriptase with random hexamer primers (Invitrogen). Gene expression was assessed using standard qPCR approaches with either Power Sybr Green or Taqman Master Mix (Applied Biosystems). Analysis was performed on a 7900HT Fast Real-Time PCR Machine (Applied Biosystems) with the following Sybr primers: Myomaker-F: 5’-ATCGCTACCAAGAGGCGTT-3’, Myomaker-R: 5’-CACAGCACAGACAAACCAGG-3’. Taqman probes for Myogenin, MyoD, Ckm, and Myh4 were purchased from Applied Biosystems. Expression levels were normalized to 18S and represented as fold change.
In situ hybridizations
For whole mount in situ hybridization, embryos were fixed overnight in 4% PFA/PBS at 4°C, then dehydrated in increasing concentrations of methanol and bleached with 6% H202/methanol for 1 hour. Embryos were subsequently rehydrated, treated with proteinase K, and fixed in 4% PFA, 0.2% glutaraldehyde for 20 minutes. Pre-hybridization (50% Formamide, 5× SSC pH 4.5, 2%SDS, 2% blocking reagent (Roche), 250 µg/ml tRNA, 100 µg/ml heparin) was achieved at 70°C for 1 hour followed by incubation with digoxigenin-labeled probe overnight. Embryos were first washed with Solution 1 (50% Formamide, 2× SSC pH 4.5, and 1% SDS) three times, 6 times in Solution 2 (100 mM Maleic Acid, 150 mM NaCl, 0.1% Tween-20, pH 7.5), then blocked with consecutive 1 hour incubations with 2% blocking reagent/Solution 2 and 2% blocking reagent/20% heat-inactivated goat serum/Solution 2. To detect bound probe we performed immunohistochemistry with anti-digoxigenin-Alkaline Phosphatase antibody (1:2000, Roche). To develop the AP signal, embryos were washed with Solution 1, then incubated with Solution 4 (100 mM NaCl, 100 mM Tris-Cl, pH 9.5, 50 mM MgCl2, 0.1% Tween-20) with developing reagents (0.25 mg/ml NBT (Nitro blue tetrazolium chloride), and 0.125 mg/ml BCIP (5-Bromo-4-chloro-3-indolyl phosphate, toluidine salt, Roche). Lastly, the embryos were washed with Solution 4, fixed in 4% PFA/PBS at 4°C overnight, and imaged with a Zeiss 11 Stereoscope. Full length coding sequence was used to generate probes for both MyoD and Myomaker by using the digoxigenin labeling kit (Roche) followed by purification with MicroSpin™ G-25 columns (Amersham).
Radioisotopic in situ hybridization was performed as previously described32
. Briefly, sections were deparaffinized, permeabilized, and acetylated prior to hybridization at 55°C with riboprobes diluted in a mixture containing 50% formamide, 0.3M NaCl, 20mM Tris-HCl, pH 8.0, 5mM EDTA, pH 8.0, 10mM NaPO4
, pH 8.0, 10% dextran sulfate, 1× Denhardt’s, and 0.5mg/ml tRNA. Following hybridization, the sections were rinsed with increasing stringency washes, subjected to RNAse A (2µg/ml, 30min at 37°C) and dehydrated prior to dipping in K.5 nuclear emulsion gel (Ilford, UK). Autoradiographic exposure ranged from 21 to 28 days. The myogenin probe corresponded to nucleotides 31 through 638 of the coding sequence, whereas nucleotides 181–811 of the coding sequence was used for the M-cadherin probe. The Myomaker probe was full-length coding sequence. 35
S-labeled sense and antisense probes were generated by Sp6 and T7 RNA polymerases, respectively, from linearized cDNA templates by in-vitro transcription using the Maxiscript kit (Ambion).
Cardiotoxin (CTX) from Naja mossambica mossambica (Sigma) was dissolved in sterile saline to a final concentration of 10 µM and aliquoted and stored at –20°C. Mice were anesthetized by intraperitoneal injection of 2.5% Avertin at (15 µl/g). Mouse legs were shaved and cleaned with alcohol. Tibialis anterior (TA) muscles were injected with 50 µl of CTX with a 26-gauge needle.
For whole-mount X-gal staining, either embryos or tissues were fixed in 4% PFA/PBS (containing 0.01% deoxycholic acid and 0.02% Igepal) for 45 minutes at 4°C with gentle shaking then rinsed 2 times with cold PBS. Samples were stained overnight in staining solution (5mM K3Fe(CN)6, 5mM K4Fe(CN)6, 2mM MgCl2, 1mg/ml X-gal in PBS) followed by washing twice in PBS and post-fixing with 4% PFA/PBS.
For X-gal staining of cryosections or cells in culture the following procedure was employed: fix with 2% gluraraldehyde/PBS, wash 3 times in 0.1% sodium deoxycholate, 0.2% NP40 Substitute (Fluka), PBS, and incubate in staining solution (4mM K3Fe(CN)6, 4mM K4Fe(CN)6, 0.4mM MgCl2, 1mg/ml X-gal, 0.1% sodium deoxycholate, 0.2% NP40 Substitute in PBS) at 37°C overnight in the dark. The samples were then rinsed in PBS and fixed in 4% PFA/PBS for at least 20 minutes. Tissue sections were co-stained with light eosin, dehydrated, and mounted with Permount (Fisher). Cells were co-stained with nuclear fast red (Sigma).
Northern blot analysis
Total RNA was extracted as previously described. Fifteen micrograms of RNA was extracted, resolved on a 1% agarose/MOPS (0.2M MOPS pH 7.0, 20 mM sodium acetate, 10 mM EDTA pH 8.0) gel, and transferred to Hybond N+ membrane (Amersham). The membrane was then incubated in hybridization buffer (1% crystalline BSA (fraction V), 1mM EDTA, 0.5M NaHPO4, 7% SDS) for at least 2 hours at 68°C followed by overnight incubation with probes labeled with [α-32P]dCTP using the RadPrime DNA Labeling System (Invitrogen). Myomaker probe was generated from full-length coding sequence. The next day the membrane was washed with 1× SSC, 0.1% SDS for 10 minutes at room temperature followed by 3 washes at 68°C with 0.5× SSC, 0.1% SDS. The membrane was exposed to film at −80°C overnight and developed with a SRX101A Tabletop X-Ray Film Processor (Konica Minolta).
Histology and immunohistochemistry
For cryosections, skeletal muscle or limbs were dissected, embedded in gum tragacanth (1% in PBS), and frozen in 2-methylbutane cooled liquid nitrogen. For paraffin sections, tissue was fixed in 10% neutral buffered formalin and processed for routine paraffin histology. Frozen and paraffin sections were cut and stained with H&E using routine procedures. Immunohistochemistry was performed by fixation with 1%PFA/PBS, permeabilization with 0.2% Triton X-100 in PBS, blocking with PBS/1% BSA, 1% heat inactivated goat serum, 0.025% Tween20, incubation with primary antibody for at least 2 hours, incubation with secondary Alexa-Fluor antibodies (Invitrogen) for 1 hour, and mounting with VectaShield containing DAPI (Vector Laboratories). Anti-mouse myosin (my32, Sigma) and desmin (DAKO) antibodies were used at 1:100. The TUNEL (Invitrogen) reaction was performed exactly as described by the manufacturer. Slides were visualized using a Leica DM RXE microscope.
Isolation of primary myoblasts and immunocytochemistry
Limbs were dissected from E15 to E17.5 embryos and dissociated in 0.05% Collagenase D (Roche) in PBS at 37°C for 2–3 hrs. Ten milliliters of culture media (20% FBS/Ham F10) was added to the suspension and triturated followed by centrifugation at 1500 × g for 10 minutes at 4°C. The pellet was resuspended in 10 ml of growth media (20% FBS/Ham F10 + 2.5 ng/ml bFGF (Promega)), filtered through a 100 µm cell strainer, and plated on a 10 cm laminin coated culture dish. To enrich for myoblasts, cultures were incubated in a small volume of PBS, and the myoblasts were dislodged by knocking the plate lightly. To induce myogenesis, the cultures were placed in differentiation media (2% horse serum, DMEM) for 3–5 days. Immunocytochemistry was performed by fixing with 4% PFA/PBS, permabilization with 0.2% Triton X-100 in PBS, blocking with 3% BSA/PBS, incubation with primary antibody for at least 2 hours, then incubation with Alexa-Fluor secondary antibodies for 1 hour. Myosin antibody, used as described above, M2 Flag antibody (Sigma) at 1:500, BrdU (Roche) at 1:100, EEA1 (generous gift of Schmid Lab, University of Texas-Southwestern) at 1:500, GM130 (BD Pharmingen) at 1:300, cyclophilin D (Abcam) at 1:200, PDI (Cell Signaling) at 1:500. Cultures were co-stained with Phalloidin-rhodamine (Invitrogen) at 1:200 and nuclei were stained with Hoechst (Invitrogen). For staining of live cells, we first washed the cells with PBS and incubated in blocking buffer (3% BSA/PBS) for 15 minutes. Primary antibody incubation was then performed on ice, followed by fixation with 4% PFA/PBS, and incubation with secondary antibody. These cultures were visualized on a Zeiss LSM 780 Confocal Microscope or a Nikon Eclipse Ti Fluorescent Microscope.
Cloning, generation of retroviruses, and C2C12 infection
We cloned Myomaker coding sequence from P0 WT tongue cDNA using the following primers: Myomaker-F: 5’-ATGGGGACAGTTGTAGCCAA-3’, Myomaker-R: 5’-TCAGACACAAGTGCAGCAGA-3’. Myomaker-Flag was generated by independently cloning the regions immediately upstream (5’ PCR product) and downstream (3’ PCR product) of the site of Flag insertion. These products were used as templates, and Myomaker-F and Myomaker-R as primers, in a standard PCR sewing reaction to generate full-length Myomaker-Flag.
Retroviral plasmid DNA was generated by subcloning Myomaker and Flag-tagged Myomaker cDNA into the retroviral vector pBabe-X31
. GFP and dsRed retrovirus have been described previously32
. Ten micrograms of retroviral plasmid DNA was transfected using FuGENE 6 (Roche) into Platinum E cells (Cell Biolabs) which were plated on a 10 cm culture dish at a density of 3 × 106
cells per dish, 24 hours before transfection. Forty-eight hours after transfection, viral media was collected, filtered through a 0.45 µm cellulose syringe filter, and mixed with polybrene (Sigma) at a final concentration of 6 µg/ml. C2C12 myoblasts (obtained from ATCC) were plated on 35 mm culture dishes at a density of 3 × 105
cells/dish 24 hours prior to infection with viral media. Eighteen hours after infection, virus was removed, cells were washed with PBS, and replaced with differentiation media. These cultures were assayed between 1 and 5 days of differentiation. The actin inhibitors Cytochalasin D (Sigma) and lantrunculin B (Sigma) were used at a concentration of 0.3 µM and 0.1 µM, respectively.
Subcellular fractionation and western blot analysis
To fractionate C2C12 cells into cytosol and membrane fractions, we first washed a 10 cm dish with cold PBS and lysed the cells by dounce homogenation in hypotonic buffer (10 mM Tris pH 8.0, 1 mM EDTA). The homgenate was centrifuged at 500 × g for 5 min. to pellet nuclei and cell debris. The supernatant was centrifuged at 100,000 × g for 20 min to pellet membrane structures. The supernatant from this step was the cytosol fraction and the membrane fraction was solubilized in an equal volume of hypotonic buffer + 1% n-Dodecyl β-D-maltoside (DDM, Sigma) for further analyses by immunoblotting. For analysis of whole cell extracts, DDM solubilization was used (20 mM HEPES, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% DDM). For immunoblotting, equal protein amounts were separated on a 12% SDS-PAGE, transferred to a PVDF membrane (Millipore), blocked in 5% milk in TBS-tween and incubated with primary antibodies. The following antibodies were used: M2 Flag (Sigma, 1:1000), Gapdh (Millipore, 1:10000), VDAC (Santa Cruz, 1:1000), α-tubulin (Sigma, 1:1000), myosin (my32, Sigma, 1:1000), and myogenin (Developmental Studies Hybridoma Bank, 1:1000).
WT myoblasts were mixed with either Myomaker+/−
myoblasts at equal ratios (approximately 1 × 105
cells per genotype), plated on a well of a laminin coated 12-well plate, and induced to differentiate the next day. 10T1/2 fibroblasts were infected with either GFP- and empty-retrovirus or GFP- and Myomaker-retrovirus for 18h. After infection, cells were washed multiple times and then trypsinized, and mixed with C2C12 myoblasts at a 1:1 ratio (1 × 105
of each cell type) and plated on one well of a 6 well plate in differentiation media. GFP and myosin expression was analyzed 4 days after differentiation. A similar protocol was performed to assess incorporation of BrdU-labeled fibroblasts into myotubes with minor modifications. 10T1/2 fibroblasts were incubated with BrdU (Roche) at a final concentration of 10 µM for 18 hours. They were then infected with either empty-retrovirus or Myomaker-retrovirus and mixed with C2C12 myoblasts that had been infected with dsRed-retrovirus.
In Movie 1, C2C12 myoblasts were infected with GFP and Myomaker retrovirus. For Movie 2, C2C12 myoblasts were infected with dsRed retrovirus and fibroblasts were infected with GFP and Myomaker retrovirus. GFP and dsRED was visualized using a Perkin Elmer Ultraview Spinning Disk Confocal Microscope with a chamber for control of temperature and CO2. Images were captured every 15 minutes using Volocity 5.4.0 software. Images were analyzed and movies assembled using ImageJ.
Quantitation and statistics
Each histological analysis of embryonic skeletal muscle was performed on four samples per genotype. The differentiation index was calculated as the percentage of nuclei in myosin-positive cells. The fusion index was calculated as the percentage of nuclei contained in myosin-positive myotubes. Structures must contain at least 2 nuclei to be considered a myotube. To quantitate fusion between WT myoblasts and either Myomaker+/− or Myomaker−/− myoblasts, we calculated the percentage of LacZ+ myotubes containing ≥3 nuclei. To quantitate fusion between fibroblasts and myoblasts we calculated the percentage of GFP+ myosin+ cells or the percentage of BrdU+ myotube nuclei. For each quantitation, at least 3 independent experiments were performed in duplicate and at least 6 random fields were imaged per sample. Data are presented as mean ± SEM. Differences between groups were tested for statistical significance using the unpaired two-tailed Student’s t test. P < 0.05 was considered significant.